Intestinal disorders such as inflammatory bowel disease (IBD) and cancer frequently reflect dysregulated activity of crypt stem and progenitor cells. Relationships among these cells are presently coming into focus but it remains unclear how the key cell populations regulate lineage-specific genes to choose among alternative enterocyte and secretory cell fates. Intestinal crypt homeostasis most notably requires Notch and Wnt signaling, among other pathways. The two canonical signals cooperate to promote cell replication, but whereas Wnt signaling promotes intestinal secretory cell differentiation, Notch signaling favors enterocyte differentiation at the expense of secretory cells; the basis for these differences is unknown. Notch inhibits expression of transcription factor Atoh1 (Math1), whose activity or absence seems sufficient to allocate cell lineages by the evolutionarily conserved process of lateral inhibition. Manipulation of GI signaling pathways for clinical application will require a deeper appreciation of underlying principles and mechanisms to identify therapeutic targets that will least disrupt normal digestive physiology. This proposal applies innovative methods to isolate mouse intestinal crypt progenitors and to study in fresh detail the epigenetic and transcriptional basis of cell fate decisions. In Specific Aim 1 we will identify lineage- restricted enhancer elements and genome-wide binding sites for Atoh1 in purified secretory progenitors, with the view to determine mechanisms for its potent ability to specify the full secretory lineage. We will analyze Atoh1 occupancy at cis-elements with respect to nucleosome-histone dynamics in the transition from stem cells to committed progenitors. We will define Atoh1 target genes and investigate the roles of such genes in human pathology specimens showing aberrant secretory differentiation, including IBD samples and mucinous/signet-ring colorectal cancer. To explain the multi-faceted relationship of Wnt and Notch signaling in gut epithelial progenitors, we will test the hypothesis that Atoh1 steers the Wnt-effector transcription factor Tcf4 away from cis-elements of proliferative genes and toward those of a Wnt- dependent secretory cell program. In Specific Aim 2 we propose to isolate the earliest stem-cell progeny, bipotential progenitors able to differentiate into secretory cells or enterocytes. We will study the epigenetic basis and Atoh1 dependencies for their subsequent commitment to a single fate. We will test the hypothesis that Atoh1 controls histone marks and nucleosome dynamics not only to activate secretory cis- elements but also to repress enterocyte genes. Using genetic mouse models and up-to-date approaches in epigenomic and transcription factor analysis, we propose experiments that will give clear answers and test specific predictions and hypotheses. These studies collectively address fundamental questions, explore developmental signaling and regulatory mechanisms in molecular detail in vivo, test specific and cogent hypotheses, and will contribute toward improved understanding and treatment of common GI disorders.